Multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE) are believed to be initiated by T cell-mediated immune responses to myelin antigens. In recent years, however, a significant body of evidence has been compiled indicating the contribution of various cell populations within the central nervous system (CNS), such as microglia and astrocytes, to the development and progression of the disease. Nevertheless, the role of these cell types is far from being clearly understood. Chronic neuroinflammation and demyelination may also contribute to disease progression and chronic neurological deficits. In all these processes, in MS as well as in many other neurodegenerative diseases, astrocytes have been demonstrated to play an active role. Astrocytes respond to injury by becoming "reactive" or "gliotic", a complex cellular response whose functional significance is still poorly understood. For instance, reactive astrocytes release neurotrophins essential for neuronal survival and repair, and are also responsible for the production of pro-inflammatory molecules (cytokines, chemokines, growth factors, NO etc) growth-inhibitory molecules detrimental to functional recovery. Many of the processes occurring in reactive astrocytes are regulated by NF-kB, a key modulator of inflammation and secondary injury. The studies outlined in this proposal are designed to investigate the role of astroglial NF-kB in the pathophysiology of experimental autoimmune encephalomyelitis (EAE), taking advantage of a transgenic mouse model generated in our laboratory (GFAP-IkBa-dn mice) where NF-kB is functionally inactivated in cells expressing GFAP, such as astrocytes and non-myelinating Schwann cells. Preliminary data indicate that blocking astroglial NF-kB significantly reduces disease severity, improves functional recovery following EAE and reduces neuroinflammation and demyelination. This leads us to hypothesize that reactive astrocytes significantly contribute to disease progression and development of chronic neurological deficits in EAE and MS. This hypothesis will be tested in the four specific aims outlined below. While the results generated in our transgenic mice are very promising, the studies in Aim 1 will compare our GFAP-IkBa-dn mice to two additional mouse lines (described below) to confirm that the results obtained so far in our experimental model are uniquely associated with the astrocyte-specific inhibition of the NF-kB pathway. The first mouse line (73.12xffIKKb) is obtained by breeding a GFAP-Cre line developed in Dr. Sofroniew's laboratory to a floxed (f/f) IKKb line generated in the laboratory of Dr. Michael Karin. The second mouse line (GFAPCreERT2xffIKKb) is obtained by breeding a tamoxifen inducible GFAP-Cre line (CreERT2) developed in Dr. McCarthy's lab to the same floxed (f/f) IKKb line. In Aims 2 and 3 we will use the line(s) that provides the most robust clinical improvement over the corresponding control mice to further investigate the mechanisms at the basis of the protection provided by blocking astroglial NF-kB. Specifically, studies in Aim 2 will determine if there are differences in blood brain permeability and infiltration of leukocytes in the CNS of diseased WT and mutant mice. Studies in Aim 3 will determine the mechanisms through which inhibiting astroglial NF-kB promotes an anti-inflammatory response. Studies in this aim will focus on how inhibiting astroglial NF-kB alters T and B cell responses in the spinal cord. Finally, since demyelination is a hallmark of this disease and could be modulated by neuroinflammation, studies in Aim 4 will investigate the effect of the inhibition of astroglial-NF-kB on oligodendrocyte survival and demyelination. Our experiments will not only give insights into NF-kB signaling mechanisms, but also elucidate astrocyte responses under pathological conditions. Ultimately, our goal is to determine if interfering with these responses could be beneficial a therapeutic strategy for MS and other neurological disorders. PUBLIC HEALTH RELEVANCE: The objective of this application is to better understand the role astrocytes, a non- neuronal cell found in the brain and spinal cord, play in the pathogenesis of multiple sclerosis and its animal model EAE. Understanding the pathology of this and other diseases will help in the development of more effective therapies.